Kaohsiung J Med Sci October 2006 • Vol 22 • No 10 484
Antiphospholipid syndrome (APS) is a disease char-acterized by the hypercoagulable state with clinical manifestation of venous and arterial thrombosis and fetal loss [1,2]. The diagnosis is made according to clinical findings and one of the two laboratory crite-ria including positive anticardiolipin antibody (aCL) and lupus anticoagulant (LA) [3]. Nevertheless, several observations indicate that the coagulation system is triggered, as shown by the inhibition of physiologic
anticoagulants and the inhibition of fibrinolysis [4–7]. The inhibition of both protein C activation by throm-bin and thrombomodulin and of proteolytic cleavage of factor Va and factor VIIIa by activated protein C has been observed in association with APS [8–10]. Autoantibodies to vascular heparin sulfate proteogly-can proteogly-can block the activation of natural anticoagulant antithrombin III [11–13]. In addition, there is some evi-dence of increased plasminogen activator inhibitor-1 [14] and the autoantibody on factor XII-dependent fibrinolysis [15,16].
Under resting conditions, endothelial cells exhibit the property of an anticoagulant, which keeps ves-sels patent with uncoagulated blood. After vascular damage or inflammation, endothelial cells play an important role in triggering the coagulation system.
Received: March 27, 2006 Accepted: May 26, 2006 Address correspondence and reprint requests to: Dr Wen-Chan Tsai, Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, 100 Tzyou 1stRoad, Kaohsiung 807, Taiwan.
E-mail: [email protected]
A
CTIVATION OF
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ELLS BY
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NTIPHOSPHOLIPID
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OSSIBLE
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ECHANISM
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ATIENTS WITH
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NTIPHOSPHOLIPID
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YNDROME
Pei-Pei Chen, Yu-Chih Lin, Kun-Che Wu,1Jeng-Hsien Yen, Tsan-Teng Ou, Chen-Ching Wu, Hong-Wen Liu, and Wen-Chan Tsai
Division of Rheumatology, Department of Internal Medicine, Kaohsiung Medical University Hospital, and 1Wu Kun Che Gynecologic, Obstetric and Pediatric Hospital, Kaohsiung, Taiwan.
Antiphospholipid syndrome (APS) is an antibody-mediated hypercoagulable state characterized by recurrent venous and arterial thromboembolic events. The presence of serum antibodies are collectively termed as antiphospholipid antibodies (aPL) and is the hallmark of the disease. Interest in the pathogenesis has mostly been focused on the blood coagulation factor. However, endothe-lial cells might play an important role. When stimulated, cell membrane would flip to expose negatively charged phospholipids and activation markers such as adhesive molecules may appear. We consider that these changes may play an important role in the initiation of the thrombotic process when endothelial cells encounter aPL. In this study, we incubated human umbilical vein endothelial cells (HUVECs) with IgG isolated from patients with APS and found that the HUVECs were activated by the expression of negatively charged phospholipids, as shown by high annexin V binding and negative propidium iodide staining and by an increase in the level of intracellular cell adhesion molecule-1 on the cell surface. The above findings indicate that endothelial cells can be activated on exposure to aPL and trigger the thrombotic event.
Key Words:antiphospholipid antibodies, human umbilical vein endothelial cells
Despite much attention being focused on the blood components of the coagulation system in APS patients, we think that in the presence of antiphospholipid anti-bodies (aPL), endothelial cells may show some changes to unclothe the anticoagulant state and promote the process of hypercoagulation.
M
ATERIALS ANDM
ETHODSPatients
Five patients from outpatient clinics of the Rheumato-logy Division, Kaohsiung Medical University Hospital, who fulfilled classification criteria for APS (Sapporo criteria [3]) were enrolled in the study. All patients were either positive for LA, aCL or anti-β2-glycoprotein-1 (β2-GP-1). The age of the five patients ranged from 20 to 44 years (mean, 27 years) (Table 1). This study was approved by the medical ethics committee of Kaohsiung Medical University Hospital, and all patients signed a consent form.
Purification of IgG
Immunoglobulin G (IgG) was isolated from the sera of APS patients by protein G sepharose (Amersham, Uppsala, Sweden). The protein concentration of the purified antibodies was determined by Bradford assay and sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis. The isolated IgG was further concen-trated by the use of Amicon Ultra-4 (Millipore Corp., Billerica, MA, USA) with centrifugation at 4000 g for 10 minutes. The concentrate was then filtered by 0.22μm microporous membrane (Millipore Corp.) to make it sterile.
Cells
Human umbilical vein endothelial cells (HUVECs) were isolated from the umbilical veins of normal term
deliveries as described previously [17]. Briefly, the vein was rinsed with phosphate-buffered saline (PBS) and then with Hank’s balanced salt solution (Sigma Chemical Co., St Louis, MO, USA) through 19-gauge drawing-up needles secured into the vein. Then, 0.1% collagenase Type IV (Sigma Chemical Co.) was introduced into the umbilical vein and the cord was submerged in warm cord buffer and incu-bated at 37°C in a humidified 5% CO2at atmosphere
for 15 minutes. The collagenase solution containing HUVECs was aspirated from the vein, and cen-trifuged twice at 1,000 rpm for 5 minutes. HUVECs contained in this pellet were resuspended in Iscove’s Modified Dulbecco’s Medium (Biological Industries Ltd., Kibbutz Beit Haemek, Israel) containing 10μg/mL of endothelial cell growth supplement (Upstate Biotechnology Inc., Lake Placid, NY, USA), 10% fetal calf serum (Biological Industries Ltd.), 10μg/mL of insulin (Sigma Chemical Co.), and 33mM 3-isobutyl-1-methylxanthine (Sigma Chemical Co.), 0.1% antibiotics (penicillin, streptomycillin, ampho-tericin). HUVECs were then seeded into a sterile 75 cm2culture flask (NUNC A/S, Roskilde, Denmark)
that had been coated with 0.8% gelatin (Merck, Darmstadt, Germany). The culture medium was changed every other day and the culture continued in a humidified 5% CO2 atmosphere until 80% con-fluence was reached. HUVECs were only used from passages 2–3 in this study.
Flow cytometry
HUVECs were seeded at 4˜ 105cells/mL density on 48-well plates (Costar/Corning, Acton, MA, USA) for flow cytometry. After the 6-hour treatment of APS IgG (1 mg/mL), cells were harvested and washed in PBS. After resuspension in 100μL of annexin V bind-ing buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 2.5 mmol/L CaCl2, pH 7.4; Molecular Probes Inc.,
Table 1.Clinical and laboratory data in five antiphospholipid syndrome patients
Patient Sex Age (yr) Clinical diagnosis aPL test
1 F 24 SLE, CVA (artery thrombosis) aCL(+/–), LA(+), anti-β2-GP-1(+)
2 F 31 Vasculitis, abortion aCL(+/–), LA(+), anti-β2-GP-1(+)
3 F 44 SLE, venous thrombosis aCL(+)
4 F 20 MCTD, artery thrombosis aCL(+)
5 F 30 SLE, abortion LA(+), anti-β2-GP-1(+)
aPL= antiphospholipid antibody; SLE = systemic lupus erythematosus; CVA = cerebrovascular accident; aCL = anticardiolipin anti-body; LA= lupus anticoagulant; anti-β2-GP-1 = anti-β2-glycoprotein-1; MCTD = mixed connective tissue disease.
Eugene, OR, USA), samples were stained with 2μL of annexin V–fluorescein isothiocyanate (FITC) (Strong Biotech Corp., Taiwan) together with 1 mg/mL of propidium iodide, and incubated for 15 minutes. Intracellular cell adhesion molecule-1 (ICAM-1) was detected by adding 2μL phycoerythrin (PE) conjugate ICAM-1 (CD54; BioLegend Inc., San Diego, CA, USA) for 30 minutes. Analysis was performed on a FACScan
flow cytometry (Partec GmbH, Münster, Germany) equipped with FloMax software. For each sample, 10,000 events were collected and mean fluorescence intensity (MFI) was analyzed (Table 2).
R
ESULTSChanges in cell membrane phospholipids
When HUVECs were cultured in the presence of IgG isolated from the five patients, the cell membrane expressed more negatively charged phospholipids in their outer layers, which was represented by stronger annexin V binding. In addition, the negative staining of the cells by propidium iodide indicated that the nuclear membranes were still intact (Figure 1, Table 2).Expression of adhesive molecules
To test whether the endothelial cells were activated by the antibodies, we further analyzed the expression of the adhesion molecule ICAM-1 on the cell surface when the cells were exposed to IgG isolated from the patients. All five IgGs isolated from the patients increased the surface expression of ICAM-1 (Figure 2, Table 2).
Table 2.Expression of annexin V and intracellular cell adhesion molecule-1 (ICAM-1) and cell viability of human umbilical vein endothelial cells after incubation with antiphospholipid antibody-containing immuno-globulin G purified from five patients with antiphos-pholipid syndrome
Annexin V PI ICAM-1
MFI % MFI MFI %
Untreated 1.65 32.94 0.26 0.74 17.95 Patient 1 4.81 51.60 3.31 1.40 32.96 Patient 2 7.13 67.26 0.26 5.63 66.98 Patient 3 9.65 55.36 0.24 8.70 72.08 Patient 4 6.71 52.27 0.25 13.27 52.99 Patient 5 5.86 53.74 0.26 7.56 59.87
PI= propidium iodide; MFI = mean fluorescence intensity; %= percentage of positive cells.
128 A B 0 100 101 102 103 104 0 100 101 102 103 104 Events Events
FL1-Annexin V – FL3-Propidium iodide–
64 M1 Untreated Blank Untreated Blank
Treat-1 (Light blue) Treat-2 (Purple) Treat-3 (Blue) Treat-4 (Green) Treat-5 (Yellow)
Treat-1 (Light blue) Treat-2 (Purple) Treat-3 (Blue) Treat-4 (Green) Treat-5 (Yellow)
Figure 1.Effects of antiphospholipid antibody-containing immunoglobulin G (IgG) purified from antiphospholipid syndrome (APS) patients on: (A) annexin V expression; and (B) cell viability detected by flow cytometry. (A) After incubation of human umbilical vein endothelial cells with IgG isolated from five patients with APS, the expression of negatively charged phospholipids on the cell surface increased, which was detected by increased annexin V–fluorescein isothiocyanate binding. (B) At the same time, the intact nuclear membrane, detected by propidium iodide, denotes that the cells are still alive.
D
ISCUSSIONAPS is characterized by hypercoagulation. Besides the influence of antibodies on the blood components including coagulation factors, cofactors, protein C and protein S have been reported in many papers [18,19]. We thought that antibodies might trigger the procoagulant components of endothelial cells and hence start the coagulation cascade. Our study found that when exposed to IgGs, the cell membrane began to increase the expression of negatively charged phospholipid on their outer layers. There are two possibilities when a cell has increased expression of negatively charged phospholipids on the cell mem-brane but has an intact nuclear memmem-brane: apoptosis and cell activation. When apoptosis begins, the first sign is cell membrane flipping the inner layer of phospholipid bilayers out [20]. When the condition deteriorates, the cell membrane would further dis-rupt and encircle some cytoplasmic content to pro-duce apoptotic bodies. On the other hand, when endothelial cells are activated, they express adhesive molecules for further interaction with intravascular
cells such as white blood cells and platelets [18]. In our experiment, the increased expression of ICAM-1 on HUVECs in addition to membrane flipping indicated that the cells were activated and not in an apoptotic state. However, the possibility exists that after cell activation, the endothelial cell may progress to cell death, the so-called “activation-induced cell death”. No matter what happens after cell activation, nega-tively charged phospholipids are already exposed, and the increased negative charge on the cell surface is the key factor initiating the intrinsic pathway of the coagulation system, and the high affinity of β2-GP-1 to negatively charged phospholipids is considered to be the triggering mechanism for the development of anti-β2-GP-1, which is specific for autoimmune dis-ease associated APS [21,22].
There are a variety of aPL. The epitopes of target antigens vary among different clones of antibodies. Some patients’ antibodies are reactive only with car-diolipin, while others react with reagents of Venereal Disease Research Laboratory, LA, and even with some coagulation factors [23–25]. Antibodies are not always cross-reactive. Based on high heterogeneity of antigens, it is not possible to purify these antibodies by using a single antigen such as cardiolipin. As in many other reports, we used purified IgG from APS patients as the source of aPL [26–28]. The limitation of our study is that no healthy control serum or IgG was used. In another experiment, however, we found that when IgG from patients and antiphospholipid monoclonal antibodies were compared, IgG from normal controls had no impact on endothelial cells in the expression of procoagulant-anticoagulant mole-cules such as thrombomodulin, or von Willebrand fac-tor (paper in submission). We believe that the above changes are specific.
Many surface components of endothelial cells, such as thrombomodulin, protein C, and von Willebrand factor, are involved in the balance between antico-agulation and thrombosis [29–33]. Further work must be done to clarify which factors play critical roles after the impact of antibodies on endothelial cells and, if possible, to characterize the different targets among different clones of antibodies. Above all, we conclude that apart from the alteration of blood components after reaction with antibodies, endothelial cells also take part in the initiation of thrombosis when exposed to antibodies. 128 0 100 101 102 103 104 Events FL2-ICAM-1-PE
Treat-1 (Light blue) Treat-2 (Purple) Treat-3 (Blue) Treat-4 (Green) Treat-5 (Yellow) Untreated Blank M1
Figure 2. Effect of antiphospholipid antibody-containing immunoglobulin G (IgG) purified from antiphospholipid syn-drome (APS) patients on intracellular cell adhesion molecule-1 (ICAM-1) expression of human umbilical vein endothelial cells (HUVECs) detected by flow cytometry. After incubation of HUVECs with IgG isolated from five patients with APS, the expression of adhesion molecule ICAM-1 on the cell surface increased, as detected by increased anti-ICAM-1-phycoerythrin (PE) binding. The black line is the negative control.
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